Pinosylvine synthase genes

ABSTRACT

New genes for pinosylvine synthase (&#34;pinosylvine synthase genes&#34;) have been found, which can be incorporated into the hereditary factors (the genome) of plants that generate no pinosylvine or only inadequate pinosylvine, whereby an increased resistance of these plants to pests can be brought about. Also disclosed are vectors, host organisms, and plants transformed with the new pinosylvine synthase genes.

This is a division of application Ser. No. 07/941,469, filed on Sep. 8,1992, now U.S. Pat. No. 5,391,724.

The present invention relates to new genes, isolated from plants, forpinosylvine synthase and to their use for the transformation of vectors,host organisms and plants and for generation of plants which have anincreased resistance to pests.

3,5-Dihydroxy-stilbene, which occurs in plants and has a toxic action onpests, in particular fungi, bacteria and insects, and is thereforesuitable for warding off these pests, is called pinosylvine. Thecapacity for synthesis of these substances by the plants is regarded asan important defence mechanism. Unfortunately, only few beneficialplants have the capacity to form pinosylvine or to generate it in anamount which imparts to them an adequate resistance to pests.

The use of stilbene synthase genes for generation of plants having anincreased resistance to pests is already known from EP-A-0 309 862. Aresveratrol synthase gene from peanut plants (Arachis hypogea) isdescribed specifically in this publication.

New genes for pinosylvine synthase ("pinosylvine synthase genes") havenow been found, which can be incorporated into the hereditary factors(the genome) of plants which generate no pinosylvine or only inadequatepinosylvine, whereby an increased resistance of these plants to pestscan be brought about.

Surprisingly, the new pinosylvine synthase genes have considerablybetter resistances to pests in plants than the resveratrol synthase genefrom peanut.

By pinosylvine synthase genes there are to be understood any nucleicacid (DNA) which, after transcription into RNA and translation intoprotein, causes the formation of an enzyme which has the properties of apinosylvine synthase, this nucleic acid being isolated from its naturalenvironment or integrated into a vector or contained as "foreign" DNA oras "additional" DNA in a prokaryotic or eukaryotic DNA.

By pinosylvine synthase genes there are also to be understood thosepinosylvine synthase genes which still contain, at their start and/orend, DNA sequences which do not or do not substantially impede thefunction of the genes. These DNA sequences, which are also called "geneunits", are formed, for example, by excision with restriction enzymes,since no cutting points are available for customary restriction enzymesexactly at the start and at the end of the gene. The pinosylvinesynthase genes or the gene units can also carry on their ends those DNAsequences which are in each case appropriate for their handling (forexample "linkers").

The pinosylvine synthase genes (or the gene units) can exist in the formin which they are contained in the genome of plants ("genomic" form,including sequences which do not encode pinosylvine synthase and/or donot have a regulatory action (such as introns)), or in a form whichcorresponds to the cDNA ("copy" DNA) obtainable via RNA with the aid ofreverse transcriptase/polymerase (and no longer contains introns). Thepinosylvine synthase genes can also be present in a partly or completelysynthetic form. By synthetic genes there are also understood those whichare formed by renewed joining of parts of natural genes.

DNA segments in the pinosylvine synthase genes according to theinvention (or the gene units) can be replaced by other DNA segments orDNAs which have essentially the same action.

In the present connection, by "foreign" DNA there is to be understoodthat DNA which does not occur naturally in a certain prokaryotic oreukaryotic genome, but is taken up in this genome only by interventionby man. "Additional" DNA is intended to mean that DNA which, although itoccurs naturally in the particular prokaryotic or eukaryotic genome, hasbeen taken up in this genome in an additional amount by intervention byman. One or more copies of the "foreign" DNA or "additional" DNA can beincorporated, depending on requirements and on the nature of the case inquestion.

Pinosylvine synthase which is formed in plants or plant cells with theassistance of the pinosylvine synthase genes according to the invention(or the gene units) means any enzyme which acts like pinosylvinesynthase and, in plants, increases their resistance to pests.

The preferred pinosylvine synthase genes according to the invention arecharacterised in that they hybridise with the cDNA sequence contained inthe plasmid pin 5-49 or its components or with the cDNA sequenceaccording to SEQ ID No: 1 or its components and encode pinosylvinesynthase

FIG. 1 represents the plasmid pin 5-49. The pinosylvine synthase cDNAlies on the EcoRI fragment about 1.3 kb in size. In FIG. 1, "E" denotesEcoRI; "B" denotes Bam HI; "H" denotes Hind III; and "PL" denotespolylinkers from the plasmid pT7/T3.

Pinosylvine synthase genes which are preferred according to theinvention are the pinosylvine synthase genes which occur in pine trees(Pinus sp.), particularly preferably in Pinus sylvestris, and can beisolated from these.

The pinosylvine synthase gene, the part sequence of which is present inthe form of the cDNA on the plasmid pin 5-49 (which is described belowin more detail) and the DNA sequences which have essentially the sameaction are especially preferred as the pinosylvine synthase geneaccording to the invention.

The cDNA contained on the plasmid was isolated from Pinus sylvestris. Itconsists of a sequence about 1,300 base pairs long. A part sequence of570 base pairs originates from the sequence protocol SEQ ID No: 1.

It has been found that the pinosylvine synthase genes which occur inplants (in particular pine trees and especially preferably Pinussylvestris) have wide regions of DNA sequence homology. On the basis ofthe sequence homology, the pinosylvine synthase genes according to theinvention can therefore be isolated from plants in a simple manner withthe aid of the cDNA contained on the plasmid pin 5-49 or its componentsor the sequence information according to SEQ ID No: 1 in the customarymanner using the known methods of molecular biology.

Possible plants from which pinosylvine synthase genes according to theinvention can be isolated are practically all the monocotyledonous ordicotyledonous plants, preferably dicotyledonous plants, pine trees(Pinus sp.), and particularly preferably Pinus sylvestris, beingmentioned by way of example and as preferred.

As already mentioned, pinosylvine synthase genes or the encoding regionthereof which hybridise with the cDNA which lies on the plasmid pin 5-49are preferred according to the invention. The gene or the encodingregion of the gene can be obtained in the customary manner with the aidof the cDNA.

The Escherichia coli strain E. coli pin 5-49 contains the plasmid pin5-49. This strain has been deposited at the Deutsche Sammlung vonMikroorganismen (DSM, German Collection of Microorganisms), MascheroderWeg 1b, D-3300 Braunschweig, Federal Republic of Germany, in accordancewith the conditions of the Budapest Treaty on the InternationalRecognition of Deposition of Microorganisms for the purposes of PatentProceedings (deposition date: 29th Aug. 1991). It has been givendeposition number DSM 6689.

The present invention also relates to this strain and its mutants. Theplasmid pin 5-49 deposited in this host can easily be obtained in thenecessary amounts in the customary manner by multiplication of thestrain and subsequent isolation of the plasmid.

Functionally complete genes, such as the pinosylvine synthase genesaccording to the invention, consist of a component which has aregulatory action (in particular a promoter) and the structural genewhich encodes the protein pinosylvine synthase.

Both parts of the gene can be used independently of one another. It isthus possible to follow the component having a regulatory action byanother DNA sequence (deviating from the pinosylvine synthase gene)which is to be expressed after incorporation into the plant genome.Since only relatively few isolated promoters which can display theiraction in plants or plant parts are known, the promoters of thepinosylvine synthase genes, to which the present invention likewiserelates, are useful aids in the generation of transformed plants orplant cells.

It is also possible to precede the pinosylvine synthase structural genesby a "foreign" component having a regulatory action. This could beadvantageous if only certain (for example endogenous to the plant) geneshaving a regulatory action can have a sufficient action in certainplants. The pinosylvine synthase structural genes are therefore usefulunits which can be used independently and, as already mentioned, thepresent invention also relates to them. The pinosylvine synthase genesaccording to the invention can be separated into the components having aregulatory action and the structural genes by the customary methods. Itis also possible to combine components of different naturally occurringpinosylvine synthase genes to give new functional "synthetic" genes. Thecomplete natural pinosylvine synthase genes according to the invention(or the gene units) are preferably used.

It is possible, with the aid of customary methods, to incorporate thepinosylvine synthase genes (or the gene units) or components thereof oneor several times (for example tandem arrangement), preferably once, intoany desired prokaryotic (preferably bacterial) or eukaryotic (preferablyplant) DNA as "foreign" or "additional" DNA. Thus, for example, theprotein encoding DNA can be provided with regulatory sequences andincorporated into plants. The present invention relates to therecombinant DNA "modified" in this way, which can be used, for example,for the transformation of plants or plant cells and is contained inplants or plant cells after the transformation.

The pinosylvine synthase genes (or the gene units) and/or theircomponents and the recombinant DNA can be contained, as "foreign" or"additional" DNA in vectors (in particular plasmids, cosmids or phages),in transformed microorganisms (preferably bacteria, in particularGram-negative bacteria, such as E. coli) and in transformed plant cellsand plants or in the DNA thereof. The present invention relates to suchvectors, transformed microorganisms (which can also contain thesevectors) and the transformed plant cells and plants and DNA thereof.

As already indicated, according to the invention the pinosylvinesynthase genes (or the gene units) are incorporated once or severaltimes (at the same or different points of the genome) into the naturalplant genome, it also being possible for different genes to be combinedwith one another. In the case of plants which already have the capacityfor pinosylvine synthase synthesis, the incorporation of one or morepinosylvine synthase genes according to the invention can lead toconsiderably improved resistance properties. In the case of plants whichcontain no pinosylvine synthase genes, an increased resistance to pestsis likewise achieved by incorporation of such genes. If appropriate,only the structural genes according to the invention are used, thesebeing preceded by any regulatory DNA element which may have beenisolated from the particular plant.

The increased resistance of the transformed plant cells and plantsaccording to the invention is of importance for agriculture and forestsand for cultivation of ornamental plants, cultivation of medicinalplants and plant breeding. It is also advantageous in the culture ofplant cells, for example for the production of pharmaceutically usablesubstances, to have available plant cells which have increasedresistances to attack by microbial pests, in particular fungi.

The present invention thus also relates to a process for the preparationof transgenic plant cells (including protoplasts) and plants (includingplant parts and seeds) having an increased resistance to pests, which ischaracterised in that

(a) one or more pinosylvine synthase genes (or gene units) and/orcomponents of the pinosylvine synthase genes (or of the gene units)and/or recombinant DNA according to the invention are inserted into thegenome of plant cells (including protoplasts), and if appropriate

(b) complete transformed plants are regenerated from the transformedplant cells (including protoplasts) and if appropriate propagated, andif appropriate

(c) the desired plant parts (including seeds) are obtained from theresulting transgenic plants of the parent generation or furthergenerations obtained therefrom.

Process steps (a), (b) and (c) can be carried out in the customarymanner by known processes and methods.

The present invention also relates to transgenic plant cells (includingprotoplasts) and plants (including plant parts and seeds) which containone or more pinosylvine synthase genes (or gene units) and/or componentsof the pinosylvine synthase genes (or of the gene units) as "foreign" or"additional" DNA, and to those transformed plant cells and plants whichare obtainable by the above processes.

The present invention also relates to the:

(a) use of the pinosylvine synthase genes (or of the gene units) and/ortheir components and/or the recombinant DNA according to the inventionand/or the recombinant vectors according to the invention and/or thetransformed microorganisms according to the invention for thetransformation of plant cells (including protoplasts) and plants(including plant parts and seeds), the

(b) use of the transgenic plant cells (including protoplasts) and plants(including plant parts and seeds) according to the invention for thegeneration of propagation material and for the generation of new plantsand propagation material thereof, the

(c) use of the pinosylvine synthase genes according to the invention (orof the gene units) and/or their components and/or the recombinant DNAaccording to the invention for combating pests and the

(d) use of the cDNA contained on the plasmid pin 5-49 or its componentsand of the DNA sequences corresponding to the sequence informationaccording to sequence protocol SEQ ID NO:1 for isolation of pinosylvinesynthase genes or components thereof from plants and for thedetermination of pinosylvine synthase genes in plants and (generally) inthe generation of transgenic plant cells (including protoplasts) andplants (including plant parts and seeds).

There are a number of different methods available for inserting thepinosylvine synthase genes or the gene units or their components intothe genetic material of plants or plant cells as "foreign" or"additional" DNA. The gene transfer can be carried out by the generallycustomary known methods, the expert being able to determine withoutdifficulty the particular method suitable.

The Ti plasmid from Agrobacterium tumefaciens is available as aparticularly favorable and widely applicable vector for the transfer offoreign DNA into genomes of dicotyledonous and monocotyledonous plants.The genetic material which encodes pinosylvine synthase is inserted intothe T-DNA of suitable Ti plasmids together with regulatory DNA sequences(for example Zambryski et al. 1983) and transferred by infection of theplants, infection of plant parts or plant tissues, such as, for example,of leaves, stems, hypocotyls, cotyledons, meristems and tissues issuingtherefrom, such as, for example, secondary embryos and calli, or bycoculture of protoplasts with Agrobacterium tumefaciens.

An alternative is the incubation of purified DNA which contains thedesired gene in plant protoplasts (for example Hain et al., 1985; Krenset al., 1982; Paszkowski et al., 1984) in the presence of polycations orcalcium salts and polyethylene glycol.

The DNA uptake can also additionally be promoted by an electrical field(electroporation) (for example Fromm et al., 1986).

The DNA can also be introduced in a known manner via plant pollen, by"shooting" the pollen with physically accelerated particles which carrythe DNA (compare EP-A 0,270,356).

The plants are regenerated in a known manner with the aid of suitablenutrient media (for example Nagy and Maliga 1976).

In a preferred embodiment of the process according to the invention (inaccordance with the method from EP-A 116,718), the genes or gene unitswhich, complementary to the cDNA from pin 5-49, are present in thegenome of Pinus, are cloned in isolated form into a suitableintermediate E. coli vector, for example pGV700 or pGV710 (compareEP-A-116,718), or preferably derivatives thereof, which additionallycontain a reporter gene, such as, for example, nptII (Herrera-Estrellaet al. 1983) or hpt (Van den Elzen et al 1986).

The plasmid constructed in this way is transferred by customary methods(for example Van Haute et al. 1983) to Agrobacterium tumefaciens, whichcontains, for example, pGV 3850 or derivatives thereof (Zambryski et al.1983). Alternatively, the pinosylvine synthase gene unit can be clonedin a binary vector, for example pCV001 or pCV002 (for example Koncz andSchell 1986) and transferred into a suitable Agrobacterium strain asdescribed above (Koncz and Schell 1986). The resulting Agrobacteriumstrain which contains the pinosylvine synthase genes or gene units in aform which can be transferred to plants is subsequently used for theplant transformation.

In another preferred embodiment, the pinosylvine synthase gene unitsisolated, if appropriate together with another plasmid which contains areporter gene for plant cells, for example for kanamycin resistance (forexample Herrera-Estrella et al. 1983) or a hygromycin resistance (vanden Elzen, 1986), preferably pLGV neo 2103 (Hain et al. 1985), pMON 129(Fraley R. T. et al., Proc. National Acad. Sci. USA 80, 4803 (1983), pAK1003, pAK 2004 (Velten J. et al., EMBO Journ. Vol. 3, 2723 (1984) orpGSST neo 3 (pGSST3) (EP-A-189,707), are transferred to plantprotoplasts in the customary manner by direct gene transfer (for exampleHain et al. 1985). In this case, the plasmid or plasmids can be presentin circular form, but preferably in linear form. If a plasmid with areporter gene is used, kanamycin-resistant protoplasts are then checkedfor expression of pinosylvine synthase. Otherwise (without a reportergene), the resulting calli are checked for expression of the pinosylvinesynthase gene or genes (screening by customary methods).

Transformed (transgenic) plants or plant cells are generated by theknown methods, for example by leaf disc transformation (for exampleHorsch et al. 1985) by coculture of regenerating plant protoplasts orcell cultures with Agrobacterium tumefaciens (for example Marton et al.1979, Hain et al. 1985) or by direct DNA transfection. Resultingtransformed plants are detected either by selection for expression ofthe reporter gene, for example by phosphorylation of kanamycin sulphatein vitro (Reiss et al. 1984; Schreier et al. 1985) or by the expressionof nopaline synthase (according to Aerts et al. 1983) or pinosylvinesynthase by Northern blot analysis and Western blot analysis. Thepinosylvine synthase and the stilbenes can also be detected in a knownmanner with the aid of specific antibodies in transformed plants.Pinosylvine synthase can also be detected by an enzyme activity test(Gehlert et al., 1990).

Culture of the transformed plant cells and regeneration to give completeplants are carried out by the generally customary methods with the aidof the particular suitable nutrient media.

Both the transformed plant cells and the transformed plants whichcontain the pinosylvine synthase genes according to the invention (orthe gene units) and to which the present invention relates exhibit aconsiderably higher resistance to pests, in particular phytopathogenicfungi.

In connection with the present invention, the term "plants" denotes bothcomplete plants and also parts of plants, such as leaves, seeds, tubers,cuttings and the like. "Plant cells" include protoplasts, cell lines,plant calli and the like. "Propagation material" denotes plants andplant cells which can be used for propagation of the transformed plantsand plant cells, and the present invention thus also relates to thismaterial.

In the present connection, the term "DNA sequences having essentiallythe same action" means that the invention also relates to thosemodifications in which the function of the pinosylvine synthase genesand their components is not impaired such that pinosylvine synthase isno longer formed or the regulatory gene component is no longer active.Corresponding modifications can be made by replacement, addition and/orremoval of DNA sections, individual codons and/or individual nucleicacids.

In the case of microorganisms which can be used according to theinvention, "mutants" denote those modified microorganisms which stillhave the features essential for implementation of the invention, and inparticular contain the plasmid pin 5-49.

The plants which can be given resistance or an increased resistance topests by incorporation (transformation) of the pinosylvine synthasegenes according to the invention (or the gene units) include practicallyall plants. There is of course a particular need for generatingresistance in crop plants, such as forest plants, for example spruce,fir, Douglas fir, pine, larch, beech and oak, as well as plants whichsupply foodstuffs and raw materials, for example cereals (in particularwheat, rye, barley, oats, millet, rice and corn), potatoes, leguminousplants (such as pulses and in particular alfalfa and soybean),vegetables (in particular cabbage varieties and tomatoes), fruit (inparticular apples, pears, cherries, grapes, citrus fruits, pineapplesand bananas), oil palms, tea, cocoa and coffee shrubs, tobacco, sisaland cotton, and in the case of medicinal plants, such as Rauwolfia andDigitalis. Potatoes, tomatoes and leguminous plants may be mentionedparticularly preferably. The pinosylvine synthase genes according to theinvention are preferably incorporated into the genome of plants as"foreign" DNA.

As pests against which resistances or increased resistances can beachieved with the aid of the pinosylvine synthase genes according to theinvention there may be mentioned animal pests, such as insects, mitesand nematodes, as well as microbial pests, such as phytopathogenicfungi, bacteria and viruses. Microbial pests, in particularphytopathogenic fungi, are particularly singled out.

The harmful insects include, in particular, insects of the orders:

Orthoptera, Dermaptera, Isoptera, Thysanoptera, Heteroptera, Homoptera,Lepidoptera, Coleoptera, Hymenoptera and Diptera.

The harmful mites include, in particular: Tarsonemus spp., Panonychusspp. and Tetranychus spp.

The harmful nematodes include, in particular: Pratylenchus spp.,Heterodera spp. and Meloidogyne spp.

The microbial pests include, in particular, the phytopathogenic fungi:

Plasmodiophoromycetes, Oomycetes, Chytridiomycetes, Zygomycetes,Ascomycetes, Basidiomycetes and Deuteromycetes.

The phytopathogenic bacteria include, in particular, thePseudomonadaceae, Rhizobiaceae, Enterobacteriaceae, Corynebacteriaceaeand Streptomycetaceae.

The virus diseases include, in particular, mosaic, dwarfing andyellowing viroses.

Some causative organisms of viral, fungal and bacterial diseases whichcome under the generic names listed above may be mentioned as examples,but not by way of limitation:

barley yellow dwarf virus (BYDV), potato virus Y (PVY), cucumber mosaicvirus (CMV), watermelon mosaic virus (WMV), Tristeza virus, tobaccomosaic virus (TMV), tobacco necrosis virus (TNV), beet necrotic yellowvein virus (BNYVV), rhizomania virus.

Xanthomonas species, such as, for example, Xanthomonas campestris pv.oryzae; Pseudomonas species, such as, for example, Pseudomonas syringaepv. lachrymans; Erwinia species, such as, for example, Erwiniaamylovora; Pythium species, such as, for example, Pythium ultimum;Phytophthora species, such as, for example, Phytophthora infestans;

Pseudoperonospora species, such as, for example, Pseudoperonosporahumuli or Pseudoperonospora cubense; Plasmopara species, such as, forexample, Plasmopara viticola; Peronospora species, such as, for example,Peronospora pisi or P. brassicae;

Erysiphe species, such as, for example, Erysiphe graminis; Sphaerothecaspecies, such as, for example, Sphaerotheca fuliginea; Podosphaeraspecies, such as, for example, Podosphaera leucotricha; Venturiaspecies, such as, for example, Venturia inaequalis; Pyrenophora species,such as, for example, Pyrenophora teres or P. graminea (conidia form:Drechslera, syn: Helminthosporium); Cochliobolus species, such as, forexample, Cochliobolus sativus (conidia form: Drechslera, syn:Helminthosporium); Uromyces species, such as, for example, Uromycesappendiculatus; Puccinia species such as, for example, Pucciniarecondita; Tilletia species, such as, for example, Tilletia caries;Ustilago species, such as, for example Ustilago nuda or Ustilago avenae;Pellicularia species, such as, for example, Pellicularia sasakii;Pyricularia species, such as, for example, Pyricularia oryzae; Fusariumspecies, such as, for example, Fusarium culmorum;

Botrytis species, such as, for example, Botrytis cinerea; Septoriaspecies, such as, for example, Septoria nodorum; Leptosphaeria species,such as, for example, Leptosphaeria nodorum; Cercospora species, suchas, for example, Cercospora canescens; Alternaria species, such as, forexample, Alternaria brassicae; and Pseudocercosporella species, such as,for example, Pseudocercosporella herpotrichoides. Helminthosporiumcarbonum may furthermore be mentioned.

The present invention shall be illustrated in more detail with the aidof the following embodiment examples:

1. Isolation of the Gene for Pinosylvine Synthase from Pinus

Plants and cell cultures from Pinus (Pinus sylvestris) contain the genesfor pinosylvine synthase which cause the formation of pinosylvinesynthase (size of the protein 45 00 D; reaction with specific antiserum)(Gehlert et al. 1990).

The known processes and methods of molecular biology such as aredescribed in detail, for example, in the following handbook were used inthe isolation of the pinosylvine synthase genes: Sambrook, J., Fritsch,E. F., Maniatis, T.: Molecular Cloning: A Laboratory Manual; Cold SpringHarbor Laboratory, Second Edition 1989.

A "gene library" for Pinus is first established: genomic DNA fromenriched cell nuclei (Bedbrook, J., Plant Molecular Biology Newsletter2, 24, 1981) is cut with the restriction enzyme NdeII such that DNAfragments having an average length of about 12,000 nucleotide pairs areformed. These fragments are cloned into the BamHI site of the lambdaphage EMBL4 (Frischauf et al., J. Mol. Biol. 170, 827-842, 1983), andthe phages are multiplied in E. coli. The phage population in itsentirety contains, cloned in part fragments, the total genomic DNA ofthe Pinus-cells, and therefore also the genes for pinosylvine synthases(multigene family).

The genes for pinosylvine synthase, their mRNA and the pinosylvinesynthase cDNA each contain the same nucleic acid sequences, since theycan be derived from one another (gene→mRNA→cDNA). This means that thegenes for pinosylvine synthase can be identified by specifichybridisation with the particular pinosylvine synthase cDNA or withspecific oligonucleotides. Using this procedure genomic phage clones forpinosylvine synthase have been identified, transferred to tobacco andfound to direct the synthesis of pinosylvine in the heterologous plants.The transgenic plants displayed an increased resistance to plantpathogens. The phages with the genes are identified by hybridisation,and then isolated and multiplied. The genomic DNA from Pinus cloned inthis phages is mapped further by analysis with various restrictionenzymes, and the position of the pinosylvine synthase genes isdetermined by further hybridisation experiments with cDNA sequences orsynthetic oligonucleotides. Finally, the gene units are cut out of thephage by digestion with restriction enzymes, cloned in thecorrespondingly cut plasmid vector pUC18 (Gibco-BRLGmbH, Eggenstein,Federal Republic of Germany) and multiplied as recombinant plasmids.

2. Description of the Plasmid Pin 5-49 (Compare FIG. 1

The plasmid consists of two components:

(i) cDNA (part sequence) of pinosylvine synthase: the cDNA which hasbeen inserted into the plasmid pT7/T3 is 1.3 kb long and can be cut outof the plasmid pin 5-49 with EcoRI.

(ii) Vector plasmid: the cDNA is cloned in the vector pT7/T3 (PharmaciaLKB GmbH, Freiburg Federal Republic of Germany). The size of the vectoris 2800 nucleotide pairs. It carries the gene for ampicillin resistance,that is to say E. coli cells with this plasmid grow in nutrient mediawhich contain the antibiotic ampicillin. Ori: designation for sequenceswhich are necessary for multiplication of the plasmid in E. coli.

The plasmid pin 5-49 carries a gene for ampicillin resistance andcontains the EcoRI fragment described above, of about 1.3 kb, aspinosylvine synthase cDNA. It can be multiplied in the customary mannerin E. coli cells which contain pin 5-49 (E. coli pin 5-49).

Preferred nutrient medium for E. coli cells (for example JA221,Nakamura, K., Inouye, M., EMBO J. 1, 771-775, 1982) which contain pin5-49 (E. coli pin 5-49):

    ______________________________________                                        Bacto-Peptone*            10 g                                                yeast extract              5 g                                                NaCl                       5 g                                                agar                      20 g                                                H.sub.2 O                  1 l                                                pH 7.5                                                                        Fermentation: 37° C., aerobic                                          ______________________________________                                         *(Bacto is a trademark of DIFCO Lab. Detroit, USA).                      

3. Transformation of Tobacco

a) Culture of tobacco shoots and isolation of tobacco protoplasts:

Nicotiana tabacum (Petit Havana SR1) is propagated as a sterile shootculture on hormone-free LS medium (Linsmaier and Skoog 1965). Shootsections are transferred to fresh LS medium at intervals of about 6-8weeks. The shoot cultures are kept in 12 hours of light (1000-3000 lux)in a culture room at 24°-26° C.

For the isolation of leaf protoplasts, about 2 g of leaves (about 3-5 cmlong) are cut into small pieces (0.5 cm×1 cm) with a fresh razor blade.The leaf material is incubated in 20 ml of enzyme solution consisting ofK3 medium (Nagy and Maliga 1976), 0.4M sucrose, pH 5.6, 2% of ZellulaseR10 (Serva) and 0.5% of Macerozym R10 (Serra) at room temperature for14-16 hours. The protoplasts are then separated from cell residues byfiltration over a 0.30 mm and 0.1 mm steel sieve. The filtrate iscentrifuged at 100×g for 10 minutes. During this centrifugation, intactprotoplasts float and collect in a band at the top margin of the enzymesolution. The pellet of cell residues and the enzyme solution aresuctioned off with a glass capillary. The prepurified protoplasts aremade up to 10 ml with fresh K3 medium (0.4M sucrose as an osmotic agent)and floated again. The washing medium is suctioned off and theprotoplasts are diluted to 1-2×10⁵ /ml for culture or subsequentinfection with Agrobacteria (coculture). The protoplast concentration isdetermined in a counting chamber.

b) Transformation of regenerating tobacco protoplasts by coculture withAgrobacterium tumefaciens:

The method of Marton et al. 1979 is used below, with minormodifications. The protoplasts are isolated as described and incubatedin a density of 1-2×10⁵ /ml in K3 medium (0.4M sucrose, 0.1 mg/l of NAA,0.2 ml in K3 medium (0.4M sucrose, 0.1 mg/l of NAA, 0.2 mg of kinetin)for 2 days in the dark and one to two days under weak light (500 lux) at26° C. As soon as the first divisions of the protoplasts occur, 30 μl ofan Agrobacterium suspension in minimal A (Am) medium (density about 10⁹Agrobacteria/ml) are added to 3 ml of regenerating protoplasts. Theduration of the coculture is 3-4 days at 20° C. in the dark. The tobaccocells are then introduced into 12 ml centrifuge tubes, diluted to 10 mlwith seawater (600 mOsm/kg) and pelleted at 60×g for 10 minutes. Thiswashing operation is repeated a further 1-2 times in order to remove themajority of the Agrobacteria. The cell suspension is cultured in adensity of 5×10⁴ /ml in K3 medium (0.3M sucrose) with 1 mg/l of NAA(naphthyl-1-acetic acid), 0.2 mg/l of kinetin and 500 mg/l of thecephalosporin antibiotic cefotaxime. The cell suspension is diluted withfresh K3 medium every week and the osmotic value of the medium isreduced gradually by 0.05M sucrose (about 60 mOsm/kg) per week.Selection with kanamycin (100 mg/l of kanamycin sulphate (Sigma), 660mg/g of active km) is started 2-3 weeks after the coculture in anagarose "bead type culture" (Shillito et al. 1983). Kanamycin-resistantcolonies can be distinguished from the background of retarded colonies3-4 weeks after the start of the selection.

c) Direct transformation of tobacco protoplasts with DNA. Calciumnitrate-PEG transformation.

About 10⁶ protoplasts in 180 μl of K3 medium are carefully mixed in aPetri dish with 20 μl of aqueous DNA solution which contains 0.5 μg/μlof plasmid carrying the genomic pinosylvine synthase gene and 0.5 μg/μlof pLGV neo 2103 (Hain et al. 1985). 200 μl of fusion solution (0.1Mcalcium nitrate, 0.45M mannitol, 25% of polyethylene glycol (PEG 6000),pH 9) are then carefully added. After 15 minutes, 5 ml of washingsolution (0.275M calcium nitrate, pH 6) are added, and after a further 5minutes, the protoplasts are transferred into a centrifuge tube andpelleted at 60×g. The pellet is taken up in a small amount of K3 mediumand cultured as described in the next section. Alternatively, theprotoplasts can be transformed as described by Hain et al. 1985.

The transformation can also be carried out without the addition of the0.5 μg/μl of pLGV neo 2103. Since no reporter gene is used in this case,the resulting calli are checked for the presence of the pinosylvinesynthase gene unit with the aid of a dot blot hybridisation. The cDNAsequence from pin 5-49 can be used as the hybridisation sample. Otherdetection methods, such as a test with antibodies or determination of afungus resistance, can of course also be used.

d) Culture of the protoplasts incubated with DNA and selection ofkanamycin-resistant calli:

A modified "bead type culture" technique (Shillito et al. 1983) is usedfor the culture and selection of kanamycin-resistant colonies describedbelow. One week after treatment of the protoplasts with DNA (compare c),3 ml of the cell suspension are mixed with 3 ml of K3 medium (0.3Msucrose+hormones; 1.2% (Seaplaque) of LMT agarose, Marine Colloids) in 5cm Petri dishes. For this purpose, the agarose is autoclaved in the drystate and, after addition of K3 medium, is boiled up briefly in anmicrowave oven. After the agarose has solidified, the agarose discs("beads") are transferred into 10 cm Petri dishes with the embeddedtobacco microcalli for further culture and selection, and in each case10 ml of K3 medium (0.3M sucrose, 1 mg/l of NAA, 0.2 mg/l of kinetin)and 100 mg/l of kanamycin sulphate (Sigma) are added. The liquid mediumis changed every week. During this procedure, the osmotic value of themedium is reduced in stages.

The replacement medium (K3+km) is reduced by 0.05M sucrose (about 60mOsm) per week.

    ______________________________________                                        Timetable of the selection of kanamycin-resistant                             tobacco colonies after DNA transformation:                                                                                sucrose                                                                       in the                                  0.4 M           0.25 0.20 0.15 M      liquid                            A     ES      0.3 M   M    M    K     0.10 M                                                                              medium                            ______________________________________                                        DNA   1       2       3    4    5           6 weeks                                                                       after                             ______________________________________                                        Uptake                                                                        (K3 medium 1 mg of NAA, 0.2 mg of kinetin)                                    A = DNA uptake                                                                E = embedding in agarose                                                      S = selection with kanamycin (100 mg/l of kanamycin sulphate)                 K = kanamycin-resistant colonies can be clearly distinguished                 from the background                                                       

e) Regeneration of kanamycin-resistant plants:

As soon as the kanamycin-resistant colonies have reached a diameter ofabout 0.5 cm, half of them are placed on regeneration medium (LS medium,2% of sucrose, 0.5 mg/l of benzylaminopurine BAP) and kept in theculture room in 12 h of light (3000-5000 lux) at 24° C. The other halfare propagated as a callus culture on LS medium with 1 mg/l of NAA, 0.2mg/l of kinetin, 0.1 mg/l of BAP and 100 mg/l of kanamydin sulphate.When the regenerated shoots are about 1 cm in size, they are cut off andplaced on 1/2 LS medium (1% of sucrose, 0.8% of agar), without growthregulators, for rooting. The shoots are rooted on 1/2 MS medium with 100mg/l of kanamycin sulphate and later transferred into soil.

f) Tranformation of leaf discs by Agrobacterium tumefaciens

For transformation of leaf discs (Horsch et al. 1985), leaves about 2-3cm long from sterile shoot cultures are stamped into discs of 1 cmdiameter and incubated with a suspension of appropriate Agrobacteria(about 10⁹ /ml) (compare b) in Am medium, see below) for about 5minutes. The infected pieces of leaf are kept on MS medium (see below)without hormones for 3-4 days at about 24° C. During this period,Agrobacterium grows over the pieces of leaf. The pieces of leaf are thenwashed in MS medium (0.5 mg/ml of BAP, 0.1 mg/ml of NAA) and placed onthe same medium (0.8% of agar) with 500 μg/ml of cefotaxime and 100μg/ml of kanamycin sulphate (Sigma). The medium should be renewed aftertwo weeks. Transformed shoots are visible after a further 2-3 weeks. Theregeneration of shoots should also be carried out in parallel withoutpressure of selection. The regenerated shoots must then be tested fortransformation by biological tests, for example for nopaline synthase orpinosylvine synthase activity. 1-10% of transformed shoots are obtainedin this manner.

Biochemical Detection Methods of Transformation

Detection of nopaline in plant tissues:

Nopaline is detected as follows, as described by Otten and Schilperoort(1978) and Aerts et al. (1979). 50 mg of plant material (callus orpieces of leaf) are incubated overnight in LS medium with 0.1M arginineat room temperature in an Eppendorf vessel. The plant material is thenspotted onto absorbent paper, homogenised with a glass rod in a freshEppendorf centrifuge vessel and centrifuged in an Eppendorf centrifugefor 2 minutes. 2 μl of the supernatant are transferred in a punctiformmanner onto a paper suitable for electrophoresis (Whatman 3 MM paper)(20×40 cm) and dried. The paper is impregnated with the mobile phase (5%of formic acid, 15% of acetic acid, 80% of H₂ O, pH 1.8) and subjectedto electrophoresis at 400 V for 45 minutes. Nopaline runs towards thecathode. The paper is then dried with a stream of hot air and drawnthrough phenanthrenequinone coloring agent (equal volume of 0.02% ofphenanthrenequinone in ethanol and 10% of NaOH in 60% of ethanol) in themigration direction. The dried paper is viewed under longwave UV lightand photographed. Arginine and arginine derivatives are stained afluorescent yellow with the reagent.

Neomycin phosphotransferase (NPT II) enzyme test:

NPT II activity in plant tissue is detected as follows by in situphosphorylation of kanamycin as described by Reiβ et al. (1984) andmodified by Schreier et al. (1985). 50 mg of plant tissue arehomogenised on ice in 50 μl of extraction buffer (10% of glycerol, 5% of2-mercaptoethanol, 0.1% of SDS, 0.025% of bromophenol blue, 62.5 mM TrispH 6.8), with addition of glass powder, and centrifuged for 10 minutesin an Eppendorf centrifuge at 4° C. 50 μl of the supernatant are appliedto native polyacrylamide gel (145×110×1.2 mm; separating gel: 10% ofacrylamide, 0.33% of bisacrylamide, 0.375M Tris pH 8.8, collecting gel:5% of acrylamide, 0.165% of bisacrylamide, 0.125M Tris pH 6.8) andsubjected to electrophoresis overnight at 4° C. under 60 V. As soon asthe bromophenol blue marker runs out of the gel, the gel is washed twicewith distilled water for 10 minutes and once for 30 minutes withreaction buffer (67 mM Tris-maleate, pH 7.1, 42 mM MgCl₂, 400 mMammonium chloride). The gel is placed on a glass plate of the same sizeand covered with a layer of 40 ml of 1% strength agarose in reactionbuffer which contains the substrates kanamycin sulphate (20 μg/ml) and20-200 μCi of ³² P ATP (Amersham). The sandwich gel is incubated for 30minutes at room temperature and a sheet of phosphocellulose paper P81(Whatman) is then laid over the agarose. Four layers of 3 MM filterpaper, (Whatman) and a few paper handkerchiefs are stacked on top. Thetransfer of radioactive kanamycin phosphate phosphorylated in situ ontothe P81 paper is stopped after 3-4 hours. The P81 paper is incubated for30 minutes in a solution of proteinase K and 1% of sodium dodecylsulphate (SDS) at 60° C. and then washed 3-4 times in 250 ml of 10 mMphosphate buffer pH 7.5 at 80° C., dried and autoradiographed for 1-12hours at -70° C. (XAR5 film from Kodak).

4. Transformation of Solanum tuberosum (potato)

The transformation was transformed in exactly the manner described inEP-A-0,242,246, pages 14 to 15, the Agrobacteria containing Ti plasmidswhich carry pinosylvine synthase genes.

All the percentage data in the above examples relate to percentages byweight, unless stated otherwise.

The presence of the pinosylvine synthase genes in the plant cells andplants (tobacco) obtained according to the above examples was confirmedby Southern blot analysis. The expression of the pinosylvine synthasegenes was detected by Northern blot analysis, and the pinosylvinesynthase and pinosylvine were detected with the aid of specificantibodies. Transformed and non-transformed plants (for comparison) weresprayed with a spore suspension of Botrytis cinera and the fungal attackwas rated after 1 week. The transformed plants showed an increasedresistance to fungal attack (compared with the non-transformedcomparison plants).

Hybridisation With the cDNA Sequence Contained in Plasmid Pin 5-49 orthe cDNA Sequence According to SEQ ID No. 1

As mentioned above, the preferred pinosylvine synthase genes accordingto the invention are characterised in that they hybridise with the cDNAsequence contained in the plasmid pin 5-49 or its components or with thecDNA sequence according to SEQ ID No. 1 or its components and encodepinosylvine synthase. Moreover, the hybridisation can be generally usedfor the isolation and determination of pinosylvine synthase genes, e.g.in plants or plant parts.

Preferrably phage clones containing pinosylvine synthase genes can beidentified by hybridisation under low stringent conditions with pin 5-49(or SEQ ID No. 1) resulting in a subpopulation of clones which cansubsequently be identified as pinosylvine synthase genes clones, e.g. bydirect gene transfer into plants (Hain et al 1985, 1990) and analysis ofthe transgenic plant tissue for enzymatic activity of pinosylvinesynthase or the production of pinosylvine.

As an example pinosylvine synthase genes clones were identified usingthe cDNA clone 5-49 (or SEQ ID No. 1) as a probe under standardhybridisation conditions. Hybridisation was for 12 hours at 68° C. instandard buffer containing 2 SSC. Washes were performed at 74° C. in 2SSC and 0.1% SDS (2 times, 30 minutes), followed by one wash in 0.2 SSC,0.1% SDS for 10 minutes. Phage clone DNA was cotransferred (with a plantselectable marker (kanamycin resistance) into tobacco protoplasts andfound to direct the synthesis of pinoslyvine in tobacco.

Some of the media employed in the transformation of plants and plantcells are described below:

Am medium

3.5 g of K₂ HPO₄

1.5 g of KH₂ PO₄

0.5 g of Na₃ citrate

0.1 g of MgSO₄ ×7 H₂ O

1 g of (NH₄)₂ SO₄

2 g of glucose to 1 l

Medium for Sterile Shoot Culture of Tobacco

    ______________________________________                                        Macroelements                                                                           1/2 of the concentration of the MS salts                            Microelements                                                                           1/2 of the concentration of the MS salts                            Fe-EDTA   Murashige and Skoog (MS)                                            Myo-inositol                100     mg/l                                      Sucrose                     10      mg/l                                      Agar                        8       g/l                                       Vitamins  Ca panthotenate   1       mg/l                                                Biotin            10      mg/l                                                Nicotinic acid    1       mg/l                                                Pyridoxine        1       mg/l                                                Thiamine          1       mg/l                                      pH 5.7 before autoclaving                                                     ______________________________________                                    

K3 medium

For culture of Nicotiana tabacum petit Havana SR1, Nicotiana tabacumWisconsin 38 and Nicotiana plumbaginifolia protoplasts (Nagy and Maliga,1976)

    ______________________________________                                        Macroelements                                                                              NH.sub.4 NO.sub.3                                                                          250    mg/l                                                      KNO.sub.3    2500   mg/l                                                      CaCl.sub.2.2H.sub.2 O                                                                      900    mg/l                                                      MgSO.sub.4.7H.sub.2 O                                                                      250    mg/l                                                      NaH.sub.2 PO.sub.4.1H.sub.2 O                                                              150    mg/l                                                      (NH.sub.4).sub.2 SO.sub.4                                                                  134    mg/l                                                      CaHPO.sub.4.1H.sub.2 O                                                                     50     mg/l                                         Microelements                                                                              H.sub.3 BO.sub.3                                                                           3      mg/l                                                      MnSO.sub.4.1H.sub.2 O                                                                      10     mg/l                                                      ZnSO.sub.4.4H.sub.2 O                                                                      2      mg/l                                                      KI           0.75   mg/l                                                      Na.sub.2 MoO.sub.4.2H.sub.2 O                                                              0.25   mg/l                                                      CuSO.sub.4.5H.sub.2 O                                                                      0.025  mg/l                                                      CoCl.sub.2.6H.sub.2 O                                                                      0.025  mg/l                                         Fe-EDTA      Na.sub.2 EDTA                                                                              37.2   mg/l                                                      FeSO.sub.4.7H.sub.2 O                                                                      27.8   mg/l                                         Inositol                  100    mg/l                                         Sucrose                   137    g/l                                                                           (= 0.4 M)                                    Xylose                    250    mg/l                                         Vitamins     Nicotinic acid                                                                             1      mg/l                                                      Pyridoxine   1      mg/l                                                      Thiamine     10     mg/l                                         Hormones     NAA          1.0    mg/l                                                      Kinetin      0.2    mg/l                                         pH 5.6                                                                        Sterilise filter                                                              ______________________________________                                    

Linsmaier and Skoog Medium (Linsmaier and Skoog 1965)

For culture of regenerated protoplasts and for tissue culture of tobaccotumors and callus. Linsemaier and Skoog (LS) medium is Murashige andSkoog medium (Murashige and Skoog, 1962) with the followingmodifications:

thiamine is weighed in at a higher concentration of 0.4 mg/l instead of0.1 mg/l;

glycine, pyridoxine and nicotinic acid are absent.

    ______________________________________                                        Macroelements                                                                              NH.sub.4 NO.sub.3                                                                          1650   mg/l                                                      KNO.sub.3    1900   mg/l                                                      CaCl.sub.2.2H.sub.2 O                                                                      440    mg/l                                                      MgSO.sub.4.7H.sub.2 O                                                                      370    mg/l                                                      KH.sub.2 PO.sub.4                                                                          170    mg/l                                         Microelements                                                                              H.sub.3 BO.sub.3                                                                           6.2    mg/l                                                      MnSO.sub.4.1H.sub.2 O                                                                      22.3   mg/l                                                      ZnSO.sub.4.4H.sub.2 O                                                                      8.6    mg/l                                                      KI           0.83   mg/l                                                      Na.sub.2 MoO.sub.4.2H.sub.2 O                                                              0.25   mg/l                                                      CuSO.sub.4.5H.sub.2 O                                                                      0.025  mg/l                                                      CoCl.sub.2.6H.sub.2 O                                                                      0.025  mg/l                                         Fe-EDTA      Na.sub.2 EDTA                                                                              37.2   mg/l                                                      FeSO.sub.4.7H.sub.2 O                                                                      27.8   mg/l                                         Inositol                  100    mg/l                                         Sucrose                   30     g/l                                          Agar                      8      g/l                                          Vitamins     Thiamine     0.4    mg/l                                         Hormones:    NAA          1      mg/l                                                      Kinetin      0.2    mg/l                                         pH 5.7 before autoclaving                                                     ______________________________________                                    

The following literature can be cited for transformation of plants andplant cells:

Aerts M., Jacobs M, Hernalsteens J. P., Van Montagu M., Schell J. (1983)Induction and in vitro culture of Arabidopsis thaliana crown gallrumours. Plant Sci Lett. 17:43-50

Fromm M. E., Taylor L. P., Walbot V. (1986) Stable transformation ofmaize after gene transfer by electroporation. Nature 319: 791-793

Gehlert, R., Schoppner, A., and Kindl, H. (1990) Synthase from Seedlingsof Pinus sylvestris: Purification and Induction in Response to FungalInfection. Molecular Plant-Microbe Interactions. Volume 3, No. 6, pages444-449

Hain, R., Stable, P., Czernilofsky, A. Pp., Steinbiβ, H. H.,Herrera-Estrella, L., Schell, J. (1985) Uptake, integration, expressionand genetic transmission of a selectable chimeric gene by plantprotoplasts. Molec Gen Genet 199:161-168

Herrera-Estrella L., De Block M., Messens E., Hernalsteens J. P., vanMontagu M., Schell J. (1983) EMBO J. 2: 987-995.

Horsch R. B., Fry J. E., Hoffmann N. L., Eichholtz D., Rogers S. G.,Fraley R. T. (1985) A simple and general method for transferring genesinto plants. Scinece 277: 1229-1231

Krens F. H., Molendijk L., Wullems G. J., Schilperoort R. A. (1982) invitro transformation of plant protoplasts with Ti-plasmid DNA, Nature296:72-74

Koncz C., Schell J. (1986) The promoter of T_(L) -DNA gene 5 controlsthe tissue-specific expression of chimaeric genes carried by a novaltype of Agrobacterium binary vector. Mol. Gen. Genet. (1986) 204:338-396

Linsmaier D. M., Skoog F. (1965) Organic growth factor requirements oftobacco tissue cultures. Physiol plant 18:100-127

Marton L., Wullems G. J., Molendijk L., Schilperoort P. R. (1979) Invitro transformation of cultured cells from Nicotiana tabacum byAgrobacterium tumefaciens. Nature 277: 1229-131

Murashige, T. and Skoog F. (1962) A revised medium for rapid growth andbioassay with tobacco tissue culture. Physiol. Plant. 15, 47

Nagy J. I., Maliga P. (1976) Callus induction and plant regenerationfrom mesophyll protoplasts of Nicotiana sylvestris. Z Pflanzenphysiol78:453-455

Otten L. A. B. M. , Schilperoort R. A. (1978) A rapid microscale methodfor the detection of Lysopin and Nopalin dehydrogenase activities.Biochim biophys acta 527:497-500

Paszkowski J., Shillito R. D., Saul M., Mandak V., Hohn T., Hohn B.,Potrykus I. (1984) Direct gone transfer to plants. EMBO J 3: 2717-2722

Shillito R. D., Paszkowski J. Potrykus I. (1983) Agarose plating andBead type culture technique enable and stimulate develoopment ofprotoplast-derived colonies in an number of plant species. P1 Cell Rep2:244-247

Van den Elzen P. J. M., Townsend J., Lee K. Y., Bedbrook J. R. (1985)Achimaeric resistance gene as a selectable marker in plant cells, PlantMol. Biol. 5, 299-302.

Van Haute E., Joos H., Maes M., Warren G., Van Montagu M., Schell J.(1983) Intergenic transfer and exchange recombination of restrictionfragments cloned in pBR322: a novel strategy for the reversed geneticsof Ti plasmids of /Agrobacterium tumefaciens. EMBO J 2:411-418

Velten J., Velten L., Hain R., Schell J. (1984) Isolation of a dualplant promoter fragment from the Ti plasmid of Agrobacteriumtumefaciens. EMBO J 12:2723-2730

Wullems G. J., Molendijk L., Ooms G., Schilperoort R. A. (1981)Differential expression of crown gall tumor markers in transformantsobtained after in vitro Agrobacterium tumefaciens--inducedtransformation of cell wall regenerating protoplastss derived fromNicotiana tabacum. Proc Ntl Acad Sci 78:4344-4348

Zambryski P., Joos H., Genetello C., van Montagu M., Schell J. (1983)Ti-plasmid vector for the introduction of DNA into plant cells withoutaltering their normal regeneration capacity, EMBO J 12: 2143-2150

Reiss B., Sprengel R., Will H. and Schaller H. (1984) A new sensitivemethod for qualitative and quantitative assay of neomycinphosphotransferase in crude cell tracts, GENE 1081: 211-217

Schreier P., Seftor E., Schell J. and Bohnert H. (1985) The use ofnuclear-encoded sequences to direct the light-regulated synthesis andtransport of a foreingn protein into plant chloroplasts, EMBO J Volume4, No. 1:25-32

The following published patent applications may furthermore bementioned:

EP-A 116,718

EP-A 159,418

EP-A 120,515

EP-A-120,516

EP-A-172,112

EP-A-140,556

EP-A-174,166

EP-A-122,791

EP-A-126,546

EP-A-164,597

EP-A-175,966

WO 84/02913

WO 84/02919

WO 84/02920

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 570 nucleotides                                                   (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: cDNA from mRNA                                            (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Pinus sylvestris                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AAGAATCCCGATGTGTGCGCGTTCGTGGAGGTGCCATCG39                                     LysAsnProAspValCysAlaPheValGluValProSer                                       1510                                                                          TTGGACGCACGGCAGGCCATGTTGGCTATGGAGGTGCCC78                                     LeuAspAlaArgGlnAlaMetLeuAlaMetGluValPro                                       152025                                                                        CGGCTGGCAAAAGAGGCCGCTGAAAAGGCCATTCAGGAG117                                    ArgLeuAlaLysGluAlaAlaGluLysAlaIleGlnGlu                                       3035                                                                          TGGGGGCAGTCCAAGTCTGGGATCACTCATCTCATATTT156                                    TrpGlyGlnSerLysSerGlyIleThrHisLeuIlePhe                                       404550                                                                        TGCAGCACAACGACTCCGGATCTACCTGGAGCAGACTTT195                                    CysSerThrThrThrProAspLeuProGlyAlaAspPhe                                       556065                                                                        GAGGTAGCCAAGTTGCTGGGGCTGCACCCGAGTGTGAAG234                                    GluValAlaLysLeuLeuGlyLeuHisProSerValLys                                       7075                                                                          AGAGTGGGCGTGTTCCAACATGGCTGCTTCGCCGGAGGC273                                    ArgValGlyValPheGlnHisGlyCysPheAlaGlyGly                                       808590                                                                        ACCGTTCTTCGAATGGCGAAAGACCTTGCCGAAAACAAT312                                    ThrValLeuArgMetAlaLysAspLeuAlaGluAsnAsn                                       95100                                                                         CGAGGAGCTCGGGTGCTGGTCATCTGTAGTGAAACCACC351                                    ArgGlyAlaArgValLeuValIleCysSerGluThrThr                                       105110115                                                                     GCCGTTACCTTTCGTGGACCCTCCGAGACTCACCTGGAC390                                    AlaValThrPheArgGlyProSerGluThrHisLeuAsp                                       120125130                                                                     AGCCTGGTGGGGCAAGCTCTGTTTGGCGACGGTGCTTCT429                                    SerLeuValGlyGlnAlaLeuPheGlyAspGlyAlaSer                                       135140                                                                        GCCCTCATCGTGGGAGCTGATCCCATCCCTCAAGTGGAG468                                    AlaLeuIleValGlyAlaAspProIleProGlnValGlu                                       145150155                                                                     AAGGCCTGTTTCGAAATCGTTTGGACAGCCCAGACAGTT507                                    LysAlaCysPheGluIleValTrpThrAlaGlnThrVal                                       160165                                                                        GTTCCCAACAGCGAGGGAGCCATCGGTGGGAAGGTGAGA546                                    ValProAsnSerGluGlyAlaIleGlyGlyLysValArg                                       170175180                                                                     GAGGTCGGGCTGACCTTCCAACTC570                                                   GluValGlyLeuThrPheGlnLeu                                                      185190                                                                        __________________________________________________________________________

What is claimed is:
 1. A transformed plant cell containing within itsgenome a nucleotide sequence selected from the group consisting of (i)the nucleotide sequence of the cDNA contained in plasmid pin 5-49, (ii)the nucleotide sequence of the cDNA sequence of SEQ ID No: 1 and (iii)the nucleotide sequence of a degenerate variant of (i) or (ii), whereinsaid nucleotide sequence is expressed when said transformed plant cellis exposed to fungi, said transformed plant cell having increasedresistance to said fungi as compared to an untransformed plant cell ofthe same cell type and plant species exposed to the same fungi under thesame conditions, and said increased resistance to said fungi being aresult of the expression of said nucleotide sequence.
 2. A transformedplant part containing within its genome a nucleotide sequence selectedfrom the group consisting of (i) the nucleotide sequence of the cDNAcontained in plasmid pin 5-49, (ii) the nucleotide sequence of the cDNAsequence of SEQ ID No: 1 and (iii) the nucleotide sequence of adegenerate variant of (i) or (ii), wherein said nucleotide sequence isexpressed when said transformed plant part is exposed to fungi, saidtransformed plant part having increased resistance to said fungi ascompared to an untransformed plant part of the same plant part type andplant species exposed to the same fungi under the same conditions, andsaid increased resistance to said fungi being a result of the expressionof said nucleotide sequence.
 3. A transformed whole plant containingwithin its genome a nucleotide sequence selected from the groupconsisting of (i) the nucleotide sequence of the cDNA contained inplasmid pin 5-49, (ii) the nucleotide sequence of the cDNA sequence ofSEQ ID No: 1 and (iii) the nucleotide sequence of a degenerate variantof (i) or (ii), wherein said nucleotide sequence is expressed when saidtransformed whole plant is exposed to fungi, said transformed wholeplant having increased resistance to said fungi as compared to anuntransformed whole plant of the same plant species exposed to the samefungi under the same conditions, and said increased resistance to saidfungi being a result of the expression of said nucleotide sequence.